1. Field of the Invention
The present invention relates generally to a method of amplifying a nucleic acid region of interest and, more particularly, to a method of amplifying a nucleic acid region of interest via a nested single tube PCR. The method of the invention is designed to provide a means to selectively inactivate the functionality of the outer primer or primers and to maintain amplification efficiency throughout the reaction. The development of a means to achieve efficient amplification by the outer primer followed by efficient amplification with the inner primers, in the context of a single tube nested PCR, is useful in a range of applications including, but not limited to, the diagnosis and/or monitoring of disease conditions which are characterized by specific gene sequences and the characterization or analysis of specific gene regions of interest. Still further, the method of the present invention enables quantification to be performed and not just simple detection.
2. Description of the Related Art
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The polymerase chain reaction (PCR) is a technique which is utilised to amplify specific regions of a DNA strand. This may be a single gene, just a part of a gene or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size (Cheng et al., 1994, Proc Natl Acad Sci. 91:5695-5699).
PCR, as currently practiced, requires several basic components (Sambrook and Russel, 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed.). These components are:
a DNA template which contains the region of the DNA fragment to be amplified;
primers, which are complementary to the DNA regions at the 5′ and 3′ ends of the DNA region that is to be amplified;
a DNA polymerase (e.g. Taq polymerase or another thermostable DNA polymerase with a temperature optimum at around 70° C.), used to synthesize a DNA copy of the region to be amplified; and
Deoxynucleotide triphosphates (dNTPs) from which the DNA polymerase builds the new DNA.
PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl, which are inserted into a thermal cycler. This machine heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers comprise heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation.
Accordingly, PCR is a method that allows exponential amplification of DNA sequences within a longer DNA molecule. The reaction involves a number of cycles of amplification, and in each cycle the template for each molecular reaction is either a strand of the initial DNA in the sample or a strand of DNA synthesised in a preceding cycle. Each PCR cycle involves the following steps
denaturation by heat to separate the 2 strands of the DNA duplex
hybridization of the upstream and downstream primers to their complementary sequences
extension of the primers by the DNA polymerase to produce a complementary copy of the template sequence
Typically the PCR reagents and conditions are chosen so that denaturation, hybridization and extension occur at close to maximum efficiency and as a result the amount of the desired sequence increases with each cycle by a factor of close to 2. Substantial amplification occurs by the end of the PCR eg a 30 cycle PCR will result in amplification of the original template by a factor of almost 230 (1,000,000,000). This degree of amplification facilitates detection and analysis of the amplified product.
After a number of cycles of amplification, the PCR may be terminated and the product analysed in various ways, most commonly by gel electrophoresis. When the PCR is carried out to a finite endpoint, the amount of amplified product is usually not closely related to the amount of input target DNA, and this type of PCR is rather a qualitative tool for detecting the presence or absence of a particular DNA and/or for providing sufficient target DNA for further analysis.
In order to measure messenger RNA (mRNA), the method uses reverse transcriptase to initially convert mRNA into complementary DNA (cDNA) which is then amplified by PCR and analyzed by agarose gel electrophoresis. Reverse transcription followed by end-point PCR is similarly essentially a qualitative technique.
In order to provide quantification capability, real-time PCR was developed. This procedure follows the general pattern of PCR, but the amplified DNA is quantified during each cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA and modified DNA oligonucleotide primers or probes the fluorescence of which changes during one of the steps of the PCR. Frequently, real-time polymerase chain reaction is combined with reverse transcriptase polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time or in a particular cell or tissue type.
(i) Real-Time PCR Using Dyes Binding to Double-Stranded DNA
A DNA-binding dye, such as Sybr Green, binds to all double-stranded (ds)DNA in a PCR reaction, causing increased fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity which is measured at each cycle, thus allowing DNA concentrations to be quantified.
(ii) Fluorescent Reporter Sequence Methods
A number of different methods using fluorescent reporter primers or probes have been developed and they tend to be more accurate and reliable than use of DNA binding dyes. They use one or more DNA primers or probes to quantify only the DNA to which the primer or probe hybridises. Use of a reporter probe, such as a Taqman probe, significantly increases specificity and may allow quantification even in the presence of some non-specific DNA amplification. Use of sequence-specific primers or probes allows for multiplexing—assaying for several different amplified products in the same reaction by using specific sequences or probes with different-coloured labels, provided that all targets are amplified with similar efficiency.
In terms of quantification, relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale. A threshold for increase of fluorescence above background or decrease below background (depending on the precise method) is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct.
The amount of target DNA is then determined by comparing the test results to the results produced by one or more standards. When the target DNA is genomic DNA, then a series of standards, usually 10 fold dilutions of a known amount of the target DNA, are commonly used. When the target DNA is cDNA, then one or more internal standards of the cDNA of another gene are commonly used.
A variation of traditional PCR, designed to increase the specificity of the PCR amplification, is the nested PCR reaction. In this amplification reaction, two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one (‘hemi-nesting’) or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product.
Nested PCR is conventionally performed by carrying out an initial PCR in one reaction tube, transferring an aliquot of the amplified products into a second reaction tube, and then carrying out a second PCR. This procedure has two disadvantages. It is more complex than a single PCR and, more importantly, it carries the risk of contaminating the environment with the amplified products of the first PCR, which may lead to contamination of subsequent experimental procedures. For this reason, several methods have been developed for carrying out the successive PCRs in the one reaction tube.
Carrying out two rounds of PCR in the one reaction tube involves adding the primers for the two rounds into the initial reaction mixture. The methods that have then been used for producing two sequential rounds of PCR, the first using the outer pair of primers and the second using the inner pair, include:
using different annealing temperatures for the two rounds of PCR (Kemp et al., Gene 1990, 94:223-228, Erlich et al. U.S. Pat. No. 5,314,809),
decreasing the concentration of the primers for the first round PCR (Erlich et al. U.S. Pat. No. 5,314,809)
varying the annealing times for the two rounds of PCR Grosz et al U.S. Pat. No. 5,340,728)
modifying the structure of the primers for the second round of PCR and using two different annealing temperatures during this round. (Xu Dingbang Publication CN1858219).
using a low denaturation temperature for the second round PCR (Erlich et al U.S. Pat. No. 5,314,809)
using chemically modified primers for the first round PCR and an enzyme which would progressively destroy them (Du Breuil Lastrucci U.S. Pat. No. 7,273,730)
using an initial physical separation of the reagents for the first and second round PCRs (Yourno U.S. Pat. No. 5,556,773, Ching et al. United States Patent Application 20060177844).
The principle underlying all of these methods is to produce, at some point, a rapid or gradual inhibition of the activity of the primers for the first round PCR so that ongoing amplification depends progressively on the activity of the primers for the second round PCR. However, all of these methods have disadvantages, the nature of the disadvantage depending upon the method. Their robustness varies and the reaction conditions may need to be adjusted depending on the sequence of interest which is to be amplified. Amplification may be inefficient, in some cases throughout the first round PCR and in other cases during the transition from the first round to the second round PCR. As a consequence, some of the approaches are not widely used in practice, whereas others are used only for detection and not for quantification.
In work leading up to the present invention, a single tube nested PCR method has been developed which facilitates both specificity and efficiency by providing a mechanism to selectively inactivate the functionality of the outer primer once it is no longer required, thereby enabling efficient amplification using the outer primers followed by efficient amplification using the inner primer. This has therefore enabled the development of a PCR method which is sufficiently efficient to effect quantification, as well as detection, and which provides the unique advantages of a single tube nested PCR reaction.